Cyclic nucleotides will be studied as intracellular mediators of mitogen activation of lymphocytes. Because two pulses of con A, separated by many hours, are necessary to induce T cell proliferation, this mitogen activation system can be considered as a model system in which to study the two signal hypothesis for antibody induction. Since all known effects of cyclic nucleotides are mediated via cyclic nucelotide-dependent protein kinases, these enzymes are viewed as the ultimate effectors of cyclic nucleotide action in lymphocytes. The role of cAMP as a mediator of the first and/or second mitogen signals will be studied by determining the activation of cAMP-dependent protein kinase(s). The physiologically important cyclic nucleotides are considered to be those which are bound to their receptors. Bound cyclic nucleotide will be quantitated after mitogen stimulation. Millipore filtration is used for separating free from bound cyclic nucleotide. Compartmentalization of bound cyclic nucleotides and proteins kinases will be examined using monospecific antisera raised against the cyclic nucleotides or protein kinase subunits. These antisera will be used for immunocytochemistry including bright field (peroxidase), immunofluorescent and immunoelectron microscopy.